TnpI is a site-specific recombinase (SSR) encoded by the transposon Tn4430, a member of the TN3 family from Bacillus thuringiensis. TnpI belongs to the integrase family of SSRs, also referred to as the tyrosine recombinase family, and, as such, has structural similarities to other members of the integrase family of SSRs such as λ Int (Mahillon and Lereclus, 1988, EMBO J. 7:1515–26). It has been reported that TnpI mediates recombination of DNA molecules containing two relatively long DNA regions taken from Tn4430 in Bacillus thuringiensis (Salamitou et al., 1997, Gene 202:121–26; Sanchis et al., 1997, Appl. Environ. Microbiol. 63:779–84), and it was postulated to further require a host-derived factor (Mahillon and Lereclus, supra).
Two site-specific recombinases of the tyrosine class, Cre recombinase from the Escherichia coli phage P1, and Flp recombinase from the Saccharomyces cerevisiae 2 micron episome, have opened a new dimension in the art of intentional genetic engineering in higher eukaryotes (Kilby et al., 1993, Trends Genet. 9:413–21). Both recombinases recognize specific 34 bp recombination target sequences, called RTs. In the presence of active recombinase protein, only two corresponding RTs are required for recombination. The utility of these two recombinases lies in the fact that in both purified reactions in vitro and in living systems, other trans-acting factors or DNA sequence elements are unnecessary. By selected disposition of RTs and expression of the corresponding recombinase, precise genetic rearrangements can be effected in living cells.
Although very useful, Cre and Flp recombinases may not be suitable for many genetic engineering applications. For example, Flp originates from yeast and consequently has a thermal optimum of enzyme activity at 30° C. However, it is not very efficient at 37° C. (Buchholz et al., 1996, Nucleic Acids Res. 24:4256–62), thereby limiting its applicability in genetic engineering to those hosts that grow in the appropriate temperature range. In addition, recombination reactions catalyzed by Cre and Flp are reversible, giving rise to partially rearranged DNA molecules. Other biochemical characteristics also impinge upon recombination efficiency, again potentially limiting host range (Ringrose et al., 1998, J. Mol. Biol. 284:363–84). Thus it is important to identify new recombinases so that the genetic engineer can choose an optimal recombinase for use in the desired host organism.
Furthermore, there is great potential in genetic engineering for use of two, or more, recombinases in concert (Meyers et al., 1998, Nat. Genet. 18:136–41). However this potential has not been developed, mainly due to the absence of a suitable combination of recombinases that are efficient in a given host. Although there are now more than two hundred candidate tyrosine recombinases in the databases, it is not possible to predict which candidates will be useful for genetic engineering by protein sequence similarity alone. Many of these greater than 200 candidates probably work in multi-protein complexes and require auxiliary factors for efficient recombination. Furthermore, in many cases, their RTs are not obvious and may not be short.
Thus there is a continuing need to identify new recombinases that work efficiently on short recombination targets and do not require auxiliary factors.
Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.